(a) Field of the Invention
The present invention relates to a method for stabilizing a modified protein of SEC1 (Staphylococcal enterotoxin C1) that is one of the Staphylococcal enterotoxins, and a method for producing the same in large quantity. More particularly, the present invention relates to a method of increasing stability of a modified toxin by substituting one cysteine group in an amino acid sequence of modified toxin protein with a serine group to inhibit the dimer formation.
(b) Description of the Related Art
Generally, Staphylococcal enterotoxin (SE) is a pyrogenic toxin (hereafter referred to as “PT”). This kind of toxin is typically produced by Staphylococcus aureus and Staphylococcus pyrogens, although they have been found to be produced in the cells of mammals or pathogenic bacteria and viruses. Staphylococcal PT includes SE types (A, B, C1, C2, C3, D, E, G, H), Staphylococcal pyrogenic exotoxins (SPE) A and B, toxic shock syndrome toxin (TSST-1), etc.
Streptococcal PT includes SPE A, B, C, mitogenic factor (MF) and Streptococcal super antigen (SSA). These are exoproteins and are found in Streptococci of the B, C, F and G groups. All toxins pertaining to PT are proteins of monomers and have a molecular weight of approximately 22–28 kDa, and they are very similar in their amino acid sequences. They are divided into three groups according to the homology of the amino acid sequences. The first group includes SE type B (SEB), SE type C (SEC), SPE type A (SPEA), SSA, etc. and they are 49% or more identical in their sequences. The second group is 84% or more identical in their sequences and SE type A (SEA), SE type E (SEE), SE type D (SED), SPE type C (SPEC) are of this group. The third group has a low sequence homology, and TSST-1, SPEB and PSET belong to this group. Amino acid sequences are versatile but many of the sequences showing homology are concentrated on four loci. These loci are believed to relate to common biological activities of toxins. Such common biological activities include pyrogenicity, immune response suppression, cytokine induction, proliferation of lymphocytes, superantigenicity, etc. Such biological activity plays an important role in lethal diseases such as TSS (toxin shock syndrome). In addition, a unique biological activity of SE is inducing diseases such as vomiting, diarrhea and food poisoning. The characteristics of SE that distinguish them from other PTs are sulfide bonds forming disulfide loop structures. If the amino acid sequence of these functional structures are deleted or substituted with other amino acid sequence, SE can be used as vaccines or treating agents in humans or animals.
The present invention relates to the production of a modified Staphylococcal toxin C1 for the above-mentioned purpose, and a method for stabilizing said modified toxin. The genetic sequence of modified Staphylococcal toxin C1 (SEC1) was found by Gregory A, Bohach et al. (Molecular General Genetics (1987) 209: 15–20), and then the functional structural locus of SEC1 was found from amino acid primary sequence as a result of continuous studies of Dr. Bohach et al. (Terence N. Turner et al. (1992), Infection and Immunity 60(2): 694–697, Carolyn J. Hovde et al (1994), Molecular Microbiology 13(5): 897–909, Marcy L. Hoffman et al (1994), Infection and Immunity 62(8): 3396–3407).
Then, Bohach G. A et al. prepared a modified protein (SEC1-12) by deleting amino acid sequence 94 to 106 and combining an amino acid sequence at the deleted portion. The amino acid sequence 94 to 106 is a loop portion wherein SEC1 exhibits most functions as a superantigen. Thus, the prepared modified toxin can function as a mitogen in which most biological activities are removed, and thus it can function as a vaccine which elicits non-specific cellular immune responses as well as forms antibodies for humoral immune responses
The present inventor transferred said genes to an E. coli expression vector in order to produce a toxin protein in a large quantity to obtain recombinant modified toxin therefrom. However, there was a problem in that the formation of multiple structures due to the disulfide bonds largely increased by an odd number of cysteines in modified protein.
In order to solve these problems, the present inventors largely improved the stability of a modified protein toxin by substituting cysteine groups that cause the formation of dimer with serine groups, and successfully completed the process for preparing large quantities of modified toxin C1 whose host is E. Coli. 
Accordingly, it is an object of the present invention to provide a process for preparing a Staphylococcal modified toxin C1 in a large quantity by transforming E. coli with the modified toxin whose amino acid sequence is substituted by the amino acid sequence which inhibits the formation of multiple structures, and a use thereof in a vaccine for preventing, alleviating or treating mastitis in cows.